*Purpose: Epstein-Barr virus (EBV) infects more than 90% of adults worldwide and is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Although EBV infection is typically asymptomatic in immune-competent individuals, transplant recipients are highly susceptible to EBV-associated PTLD due to the immunosuppression treatment required to prevent graft rejection. Several lines of evidence suggest that innate immune responses including Natural Killer (NK) cells are critical in host immunity to EBV. Recently, a specific NK cell subset has been demonstrated to recognize and respond to autologous B cells latently infected with EBV. This NK subset expresses NKG2A/CD94, an inhibitory receptor that recognizes HLA-E. Since EBV-infected cells induced NK cell activation despite the expression of HLA-E, we hypothesize that EBV encoded peptides play a pivotal role in the recognition and response of NKG2A+ NK cells towards latently infected EBV+ cells.

*Methods: Using in silico analysis (NetMHCpan 4.0) we generated a peptide library derived from EBV latent cycle proteins (LMPs and EBNAs) expressed during the latency III stage of EBV infection. Initial assessment resulted in a library of 61 peptides which could potentially bind to HLA-E. Using 721.174 cells as target cells, we performed a peptide stabilization assay to test the ability of individual peptides to be presented by HLA-E. Co-culture systems with NK cells as effectors were used for degranulation (CD107a) and killing assays as well as Western blots.

*Results: We identified 37 peptides that bound and induced HLA-E expression at the surface of the target cells. The CD107a assay as a readout for NK cell activity, demonstrated that HLA-E bound EBV-derived peptides were able to inhibit NK cell activation (13 peptides), or prevent NK cell inhibition via the NKG2A receptor (8 peptides). Finally, non-inhibitory peptides prevent VAV1 dephosphorylation, a key molecule in the NKG2A inhibitory pathway. We determined that peptides encoded by LMPs tended to favor NK cell degranulation (NK cell killing) while peptides from EBNAs were inhibitory.

*Conclusions: We demonstrate that EBV latent cycle proteins can encode for peptides that bind to HLA-E and promote NK cell degranulation hence the elimination of EBV latently infected B cells. NK cells are generally refractory to calcineurin-based immunosuppression and thus may be an effective cellular strategy to control EBV diseases post-transplant.

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